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Oligonucleotide-directed single-base DNA alterations in mouse embryonic stem cells

Gene Therapy volume 10, pages 24–33 (2003)Cite this article

Abstract

We have investigated the use of single-stranded oligodeoxy-nucleotides (ssODN) to produce specific single-base alterations in episomal and chromosomal DNA in mouse embryonic stem (ES) cells. Two different reporter genes, EGFP and LacZ, each with a single point mutation that inactivates reporter activity, were used. ssODN homologous to the target sequence, except for a single mismatch at the mutant base, were used to correct the mutant reporter genes. When tested in CHO-K1 cells, the ssODN showed correction rates of 0.5–1.0%, consistent with prior reports. ssODN in the antisense orientation provided higher rates of gene conversion than those in the sense orientation for both reporter genes. Nuclear extracts from mouse ES cells exhibited nearly the same correction activity as extracts from CHO-K1 cells. ssODN corrected the mutant bases of both episomal and chromosomal mutant reporter genes in mouse ES cells. Although the efficiency of gene correction observed in ES cells is low, approximately 10−4, these results demonstrate that ssODN can produce single-base alterations in the genomic DNA of mouse ES cells. As conversion efficiency is improved by the continued development of oligonucleotide structure and DNA delivery methods, ssODN could be used to produce ES cells with specific mutations in any gene in a single step. The targeted ES cells could in turn be used to create accurate mouse models of inherited diseases.

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Acknowledgements

The authors thank Suzanne Pavluk, David Pugh and Maithili Navarathnarajah for technical assistance, and Drs Jean Bennett and Edward Pugh for helpful discussions. Supported in part by grants from the National Institutes of Health (EY13776, GM61942, HL66656, AR44350), Research to Prevent Blindness, the Rosanne H Silbermann Foundation, the Mackall Foundation Trust and the FM Kirby Foundation.

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Authors and Affiliations

  1. FM Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania School of Medicine, Philadelphia, PA, USA

    E A Pierce, Q Liu & R Omarrudin

  2. Department of Dermatology and Cutaneous Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College, Philadelphia, PA, USA

    O Igoucheva & K Yoon

  3. Institute of Medicine and Engineering, University of Pennsylvania, Philadelphia, PA, USA

    H Ma & S L Diamond

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  1. E A Pierce
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Pierce, E., Liu, Q., Igoucheva, O. et al. Oligonucleotide-directed single-base DNA alterations in mouse embryonic stem cells. Gene Ther 10, 24–33 (2003). https://doi.org/10.1038/sj.gt.3301857

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